Carotenoids are pigments that are ubiquitous throughout nature and synthesized by all photosynthetic organisms, and in some heterotrophic growing bacteria and fungi. Carotenoids provide color for flowers, vegetables, insects, fish and birds. Colors of carotenoid range from yellow to red with variations of brown and purple. As precursors of vitamin A, carotenoids are fundamental components in our diet and they play additional important role in human health. Because animals are unable to synthesize carotenoid de novo, they must obtain them by dietary means. Thus, manipulation of carotenoid production and composition in plants or bacteria can provide new or improved source for carotenoids. Industrial uses of carotenoids include pharmaceuticals, food supplements, animal feed additives, and colorants in cosmetics, to mention a few.
Industrially, only a few carotenoids are used for food colors, animal feeds, pharmaceuticals, and cosmetics, despite the existence of more than 600 different carotenoids identified in nature. This is largely due to difficulties in production. Presently, most of the carotenoids used for industrial purposes are produced by chemical synthesis; however, these compounds are very difficult to make chemically (Nelis and Leenheer, Appl. Bacteriol., 70:181-191 (1991)). Natural carotenoids can either be obtained by extraction of plant material or by microbial synthesis; but, only a few plants are widely used for commercial carotenoid production and the productivity of carotenoid synthesis in these plants is relatively low. As a result, carotenoids produced from these plants are very expensive. One way to increase the productive capacity of biosynthesis would be to apply recombinant DNA technology (reviewed in Misawa and Shimada, J. Biotech., 59:169-181 (1998)). Thus, it would be desirable to produce carotenoids in non-carotenogenic bacteria and yeasts, thereby permitting control over quality, quantity, and selection of the most suitable and efficient producer organisms. The latter is especially important for commercial production economics (and therefore availability) to consumers.
Carotenoid ketolases are a class of enzymes that introduce keto groups to the ionone ring of the cyclic carotenoids, such as β-carotene, to produce ketocarotenoids. Examples of ketocarotenoids include astaxanthin, canthaxanthin, adonixanthin, adonirubin, echinenone, 3-hydroxyechinenone, 3′-hydroxyechinenone, 4-keto-gamma-carotene, 4-keto-rubixanthin, 4-keto-torulene, 3-hydroxy-4-keto-torulene, deoxyflexixanthin, and myxobactone. Two classes of ketolase, CrtW and CrtO, have been reported. The two classes have similar functionality yet appear to have arisen independently as they share very little sequence similarity. The CrtW is a symmetrically acting enzyme that adds keto-groups to both rings of D-carotene (Hannibal et al., J. Bacteriol., 182: 3850-3853 (2000)). Fernández-González et al. (J. of Biol. Chem., 272: 9728-9733 (1997)) reported that the CrtO ketolase enzyme from Synechocystis sp. PCC6803 adds a keto-group asymmetrically to only one of the two β-ionone rings of β-carotene.
Several examples of CrtW ketolases have been reported in variety of microorganisms including Agrobacterium aurantiacum (also known as Paracoccus sp. N81106; U.S. Pat. No. 6,150,130; Misawa et al., Biochem. Biophys. Res. Comm., 209(3):867-876 (1995); and Misawa et al., J. Bacteriol., 177(2):6575-6584 (1995)), Bradyrhizobium sp. (US Patent Publication No. 20030087337; Hannibal et al., J. Bactetiol., 182(13):3850-3853 (2000)), Brevundimonas aurantiacum (de Souza et al., WO 02/079395), Brevundimonas sp. SD212 (WO2005/118812 A1 and Nishida et al., Appl. Env. Microbiol., 71(8):42864296 (2005), Paracoccus marcusii (Harker, M. and Hirschberg, N., (GenBank® CAB56059), Alcaligenes sp. (Misawa et al., 1995 (supra)), Sphingomonas melonis DC18 (U.S. Ser. No. 11/015,433), Brevundimonas vesicularis (U.S. Ser. No. 11/015,433), and Flavobacterium sp. (U.S. Ser. No. 11/015,433).
One factor influencing the economics of recombinant microbial production of astaxanthin is the enzymatic activity when introducing keto groups to the β-ionone rings of β-carotene and the various cyclic hydroxylated intermediates involved in the production of astaxanthin (FIG. 1). Many CrtW ketolases efficiently introduce keto groups to β-carotene, forming ketocarotenoids such as canthaxanthin. Production of astaxanthin requires the addition of hydroxyl groups to the ionone rings. This is typically accomplished by coexpressing at least one CrtZ hydroylase in combination with the CrtW ketolase. Recombinant expression of crtWZ genes in host cells capable of producing β-carotene typically results in a mixture of astaxanthin and various cyclic hydroxylated intermediates (e.g., zeaxanthin and adonixanthin). However, most CrtW ketolases exhibit limited activity towards these cyclic hydroxylated carotenoid intermediates. The limited activity adversely affects astaxathin production (as measured by the percentage of astaxanthin produced relative to the total carotenoid content).
Recombinant expression of the Sphingomonas melonis DC18 CrtW ketolase in an β-carotene producing microbial host cell produced essentially 100% canthaxanthin (U.S. Ser. No. 11/015,433). However, expression of the DC18 CrtW ketolase gene in a host cell capable of producing zeaxanthin results in the limited production of astaxanthin (12-14% of the total carotenoid concentration) with the majority (about 85%) of the carotenoids in the host cell being unwanted cyclic hydroxylated carotenoid intermediates (i.e., adonixanthin and zeaxanthin).
The problem to be solved therefore is to provide CrtW ketolases characterized by improved activity for converting cyclic hydroxylated intermediates into astaxanthin.